The long-term goal of this research project is to define the specificity, mechanism, and in vivo role of the ubiquitin- dependent protein degradation system. This soluble multi-enzyme system has been best characterized from reticulocytes where it appears to play a role in the degradation of abnormal proteins and intracellular organelles during maturation. Ubiquitin is also conjugated to histone H2a, and this modification is thought to play an important role in differentiation and growth as well. Several significant hemopoetic disease states may be influenced by this system, including thallasemias, sickle cell anemia, leukemia, and other hemopoeitic cancers. The in vivo compartmentalization and accessibility of the affected cell types makes these diseases attractive candidates for biochemical intervention. This proposal is designed to provide a basic biochemical description of the specificity and mechanism of a portion of this important protein degradation system. Ubiquitin is conjugated to a large number of cellular proteins and it is these carboyl-terminal conjugates which are thought to be substrates for the proteolytic enzymes of the system. Thus, ubiquitin acts as a signal sequence which is attached to cellular proteins to mark them for degradation. While a large number of proteins are conjugated to ubiquitin in the steady-state, only a few are degraded. This suggests an alternate fate of ubiquitin-protein conjugates is deconjugation to yield the intact proteins by and enzyme which can be termed ubiquitin protein lyase. This enzymatic activity has been described and partially characterized. The role of the ubiquitin cofactor in determining this specificity will be investigated with a series of site-directed mutants of the ubiquitin sequence. The putative "proof reading" functions, ie., enzymes which metabolize carboxyl-terminal derivatives, will be purified and characterized. Finally, a variety of ubiquitin conjugates will be synthesized using an acyl azide condensation, purified, and used as substrates in this system. A variety of physical studies and monoclonal antibodies to ubiquitin will be used to define the conformation of ubiquitin in the purified synthetic conjugates.